CN111763763B - Wheat grain weight related KASP primer group and application thereof - Google Patents
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Abstract
The invention discloses a group of KASP primer groups related to wheat grain weight and application thereof, wherein the primer groups comprise a F1 primer with a nucleotide sequence shown as SEQ ID NO.1, a F2 primer with a nucleotide sequence shown as SEQ ID NO.2 and a universal primer with a nucleotide sequence shown as SEQ ID NO. 3; the KASP primer group can be used for preparing a PCR detection system for wheat grain weight screening, and can be widely applied to the field of wheat breeding.
Description
Technical Field
The application relates to the field of wheat breeding, in particular to a KASP marker related to wheat grain weight and application thereof.
Background
Wheat grain weight is an important factor influencing yield, is mainly controlled by additive effect, and has highest heritability in wheat yield factors, which can reach 59-80% (Xiaoshihe, Hokko. "improvement of wheat yield potential and quality", see: Zhuang Qiansheng.: Chinese wheat variety improvement and pedigree analysis, Beijing: Chinese agricultural Press, 2003, pp 497 one-year-round 542). Wheat grain weight is Quantitative, presents continuous variation, and is regulated by micro-effective polygene (Zhang L Y, Liu D C, Guo X L, Yang W L, Sun J Z, Wang D W, Zhang A. genomic Distribution of Quantitative trap Loci for Yield and Yield-Related peptides in Common wheat flour. journal of Integrated Plant Biology,2010,52: 996-.
There have been many reports on genetic mapping studies of wheat grain weight: mason et al investigated the Wheat Yield traits at different stages of sowing and detected 2 thousand kernel weight QTLs on the 2D and 5A chromosomes (Mason R E, Hays D B, Mondal S, Ibrahim A M H, base B R. QTL for Yield, Yield Components and cancer Temperature prediction in Wheat straw soil Condition. Euphytoica, 2013,194: 243-; li et al performed multi-environmental yield trait identification on 3 RIL populations and identified 17 thousand grain weight QTLs (Li F, Wen W, He Z, Liu J, Jin H, Cao S, Geng H, Yan J, Zhang P, Wan Y, Xia X.genome-side linking mapping of yield-related traits in three core branched polypeptide sites using high-density SNP markers, therapeutic and Applied Genetics,2018,131: 1903-. Tura et al performed Yield trait evaluations in 32 settings on a DH population, identified 27 thousand-weight QTLs, and refined location of the major site QTgw.aww-1B (Tura H, Edwards J, Gahlaut V, Garcia M, Sznajder B, Baumann U, Shahinnia F, Reynolds M, Langridge P, Balyan H S, Gupta P K, Schnurbus T, flow D.QTL Analysis and Fine Mapping of a QTL for Yield-Related traces in Wheat growth in Dry and Hot environmental. Due to the differences of mapping populations and molecular markers, the positioning results have large differences.
SNP markers are novel molecular marker technologies at present, have the characteristics of genetic stability, large quantity and Wide distribution, can be suitable for High-throughput detection, thousands of SNP markers are gathered based on gene chips such as 9K, 90K, 660K, and the like developed by the SNP markers, the quality of genetic maps is improved (variation C R, Shiaoman C, Shichen W, Bevan Emma H, Stuart S, Seifolla K, Kerrie F, Cyrille S, Brown-guide G L, Alina A. genome-Wide Comparative indexes Uncecs, Selection for expression in Hexaploid research Landrendoces and sources, Procedenting of Nature Acense of science, moving platform of growth, Water research 8062, filtration of genetic engineering 8080, mineral of biological research 8057, mineral of biological research 8080, mineral of filtration of growth of genetic engineering, mineral, growth of genetic engineering 8080, mineral of growth, growth of genetic engineering, growth of rice 8057, growth of rice, rice, 2014,12: 787-; cui F, Zhang N, Fan X, Zhang W, Zhao C, Yang L, Pan R, Chen M, Han J, Zhao X. utilization of a Wheat660k Snap Array-Derived High-Density Genetic Map for High-Resolution Mapping of a Major QTL for Kernel number. scientific Reports, 2017: 3788).
The KASP (Kompetitive Allle Specific PCR) marker specifically matching the terminal base of the primer is a molecular marker Technology developed based on SNP locus difference, can carry out accurate double-Allele judgment on SNP loci, has the characteristics of high throughput, is suitable for molecular marker detection of a large number of samples, is in accordance with Breeding selection, and has wide Application prospect in Breeding (Semagn K, Babu R, Heart S, Olsen M.Single Nucleotide Polymorphism genetic typing Using Kompetitive Allle Specific Pcr (Kasp): Overview of the Technology and Its Application in Crop improvement. molecular Breeding 2014,33: 1-14).
Ningmai No. 9 and Yangmai 158 are high-yield, high-quality and disease-resistant wheat varieties which are bred by the academy of agricultural sciences of Jiangsu province and the agricultural sciences of the Li and the lower river of Jiangsu province respectively, the wheat varieties are widely planted in the middle and lower reaches of the Yangtze river and become important breeding parents, 36 varieties and 34 varieties pass examination in the national region test of the winter wheat in the middle and lower reaches of the Yangtze river and the Huainan region test of the Jiangsu province respectively in nearly 10 years, and the genetic background materials containing the Ningmai No. 9 or Yangmai 158 are 27 and account for nearly 80 percent. At present, no report is found about molecular markers related to grain weight of Yangmai 158 and Niumai No. 9.
Disclosure of Invention
Aiming at the problems, the application provides a KASP primer group corresponding to QTL-Qtkw-4A related to grain weight of Yangmai 158 and Ningmai No. 9, and the primer group can rapidly perform grain weight related genotyping on wheat materials in the middle and lower reaches of Yangmai (the definition of the wheat area is shown in the document Chengshun and Guo Wen, Wanglong Jun, south wheat in China, Nanjing, Jiangsu scientific and technological publisher, 2012, pp 23-26) so as to accelerate the breeding process.
Specifically, the application firstly provides a group of KASP primer groups related to wheat grain weight, which comprises a primer IAAV5722(F1) with a nucleotide sequence shown as SEQ ID NO.1, a primer IAAV5722(F2) with a nucleotide sequence shown as SEQ ID NO.2 and a universal primer IAAV5722(R) with a nucleotide sequence shown as SEQ ID NO. 3.
The application also provides an application of the KASP primer group in wheat grain weight detection, which comprises the following specific steps: preparing a PCR detection system by using the KASP primer combination, carrying out PCR amplification on the wheat DNA of a sample, and taking Yangmai No. 158 and Ningmai No. 9 as a reference; then, a fluorescence analyzer is utilized to perform gene typing on a sample wheat PCR amplification product, a sample aggregated with Yangmai 158 contains G allelic variation, a sample aggregated with Ningmai No. 9 contains A allelic variation, and the grain weight of the wheat sample containing the allelic variation G is obviously higher than that of the wheat sample containing the allelic variation A. In the fluorescence detection, a fluorescence analyzer carries out genotyping on QTL-Qtkw-4A at the position of 3.75-19.75 cM of a wheat 4A chromosome, the related SNP locus is G/A, and the thousand grain weight of wheat containing allele G is obviously higher than that of wheat containing allele A. Yangmai 158 contains a dominant allelic variation G, which binds to primer F1; a is a non-dominant allelic variation and can be combined with the primer F2, so that the KASP primer group is used for distinguishing the two; further, in the above application, the wheat is preferably wheat in the middle and lower Yangtze river.
Furthermore, the total PCR reaction system is 5. mu.L, including 2 XKASP Master Mix 2.5. mu.L, KASP Assay Mix 0.07. mu.L, and 20 ng. mu.L -1 2.43. mu.L of the template DNA of (1);
wherein, the preparation method of KASP Assay Mix of each 100 mu L is as follows: mu.L of 100. mu.M F1 primer, 12. mu.L of 100. mu.M F2 primer, and 30. mu. L, ddH of 100. mu.M universal primer 2 O is complemented to 100 mu L; the F1 primer is a primer sequence obtained by adding a sequence (preferably a sequence SEQ ID NO.7) capable of combining with FAM fluorescence to the front part of a nucleotide sequence SEQ ID NO. 1; the F2 primer is a primer sequence obtained by adding a sequence (preferably the sequence is SEQ ID NO.8) capable of combining with HEX fluorescence in front of a nucleotide sequence SEQ ID NO. 2; the sequence of the universal primer is shown as SEQ ID NO. 3.
The PCR reaction program is: the first step is as follows: 15min at 94 ℃; the second step is that: reducing the temperature by 0.6 ℃ in each cycle at 94 ℃, 20s, 61-55 ℃ for 1min, and performing 10 cycles in total; the third step: a total of 26 cycles of 94 ℃, 20s, 55 ℃ and 1min were carried out.
Furthermore, in the total system of the PCR reaction, the sequence of the F1 primer is shown as SEQ ID NO. 9; the sequence of the F2 primer is shown in SEQ ID NO. 10.
The invention also provides a kit for detecting wheat grain weight, which comprises: 2 XKASP Master Mix 2.5. mu.L, KASP Assay Mix 0.07. mu.L, 20 ng. mu.L -1 2.43. mu.L of the template DNA of (1); preparation method of KASP Assay MixThe following were used: each 100. mu.L of KASP Assay Mix contained 12. mu.L of F1 primer (SEQ ID NO.9) at a concentration of 100. mu.M, 12. mu.L of F2 primer (SEQ ID NO.10) at a concentration of 100. mu.M, 30. mu.L of universal primer (SEQ ID NO.3) at 100. mu.M, and ddH was added 2 Make up to 100. mu.L of O.
The invention utilizes an illumina 90k chip to carry out genome scanning on 282 recombinant inbred lines (F2-F8 generation) constructed by Ningmai No.9 XYangma 158, construct a genetic map, and the genetic map covers 21 chromosomes; QTL positioning is carried out by combining phenotype identification data of 3 environments, and finally a grain weight QTL-Qtkw-4A derived from Yangmai 158 is detected on a 4A chromosome and further converted into a KASP marker, and the influence of the KASP marker on the thousand grain weight of wheat is verified to be remarkable, so that the method can be applied to breeding selection.
Drawings
FIG. 1 is a schematic representation of the Qtkw-4A genetic mapping;
FIG. 2 is a graph showing the amplification results of different primers in the Qtkw-4A interval.
Detailed Description
The Ningmai No.9, Yangma 158 and their recombinant inbred line populations referred to in the examples below were maintained by wheat crop research laboratories of the academy of agricultural sciences of Jiangsu province.
Examples relate to the sequence:
SEQ ID NO.1:AGCTGCAACCCATCCTCT
SEQ ID NO.2:AGCTGCAACCCATCCTCC
SEQ ID NO.3:TGTGACCTACTCGATGTTCATAAC
SEQ ID NO.4:TGGACGGATGGTTGATGGCTCG
SEQ ID NO.5:ATGGACGGATGGTTGATGGCTCA
SEQ ID NO.6:TATAAGCTACTACCGCTCCGGC
SEQ ID NO.7:GAAGGTGACCAAGTTCATGCT
SEQ ID NO.8:GAAGGTCGGAGTCAACGGATT
SEQ ID NO.9:GAAGGTGACCAAGTTCATGCTAGCTGCAACCCATCCTCT
SEQ ID NO.10:GAAGGTCGGAGTCAACGGATTAGCTGCAACCCATCCTCC
example 1 screening of thousand Kernel weight marker intervals
Construction of Recombinant Inbred Line (RIL) population by using Ningmai No.9 XYangmai 158 (F 2:8 ) Including 282 families. 2015-2016 and 2016-2017, the RIL population and its parents were planted in Liuhe base of agricultural academy of sciences of Jiangsu province for 2 consecutive growing seasons.
The general cultivation management is carried out by using an augmentation design (see the documents "Lan C, Zhang Y, Herrera-Foessel S A, Basnet B R, Huerta-Espeno J, Lagudah E S, Singh R P.identification and characteristics of heliotropy and co-located resistance location to leave and string run in broken coal mutant Sujata. thermal and Applied Genetics,2015,128:549 and 561"), 2 parents and 30 randomly selected materials are planted for 2 repetitions, the rest are planted for 1 repetition, three rows of cells are planted for 60 grains, each row is 1.6m long, and the row spacing is 0.25 m. After the seeds are mature, the seeds are harvested, and thousand seed weight is measured by using ten thousand deep seed testing machines (SC-A1, Hangzhou Zhejiang). Also, the thousand kernel weight of 139 parts of wheat high-generation Line material (which applicant named Line001-Line139) was simultaneously measured for subsequent validation.
The genotype is obtained by using an illumina 90k chip. The genetic map covers 21 chromosomes, containing 41 linkage groups, 2285 bin markers, and has a total length of 3022cM (Jiang P, Zhang X, Wu L, He Y, Zhuang W, Cheng X, Ge W, Ma H, Kong L.A Novel QTL on Chromosome 5al of Yangmai 158Increases Resistance to Fusarium Head light in wheat plant Pathology 2020,69: 249-258). QTL positioning (Meng L, Li H, Zhang L, Wang J. QTL. Ichim. Mapping for Genetic Linkage Map Construction and Quantitative trap Mapping in Bipartmental positions. the Crop Journal,2015,3: 269) was performed by using an infinite interval Mapping (ICIM) of Ichim Mapping 4.1 Software, walking step was set to 0.1cM, and LOD threshold was set to 2.5.
A stable grain weight QTL-Qtkw-4A (figure 1) is detected through QTL positioning, and in both environments of 2015-2016 and 2016-2017, the stable grain weight QTL-Qtkw-4A is detected to be positioned at the 3.75-19.75 cM position of a 4A chromosome (the position is not reported to be related to the thousand grain weight QTL), the corresponding chromosome interval is BS00066066_ 51-BS 00066891_51, the physical interval is 661-683 Mb, and the dominant allelic variation of the increase of the thousand grain weight is derived from Yangmai 158.
Example 2 KASP molecular marker screening and validation
To better utilize the Qtkw-4A locus obtained by screening in example 1 for breeding, KASP markers suitable for high-throughput typing were developed based on their interval marker sequences. Designing PCR amplification primers according to the SNP sites and the flanking sequences, and developing KASP molecular markers.
KASP primer design was performed using Polymarker (http:// www.polymarker.info /), and the primers were synthesized by Biotechnology engineering (Shanghai) Inc. Each marker is provided with 2 SNP specific primers (F1/F2) and one universal primer (R), wherein a specific sequence capable of being combined with FAM fluorescence (SEQ ID NO.7: 5'-GAAGGTGACCAAGTTCATGCT-3') is added in front of the F1 primer, and a specific sequence capable of being combined with HEX fluorescence (SEQ ID NO.8: 5'-GAAGGTCGGAGTCAACGGATT-3') is added in front of the F2 primer. The primer design of 2 SNP markers having low homology selected from the multiple SNP sites in the marker region is shown in Table 1:
TABLE 1 KASP primer set
From the RILs Population, 46 fractions of material were randomly selected for sampling of young leaves, and genomic DNA was extracted by the CTAB method (see "Saghai-Maroof M A, Soliman K M, Jorgensen R A, Allard R W. Ribosol DNA Spacer-Length macromolecules in Bar: Mendelian Inheritance, Chromosomal Location, and position dynamics. proceedings of the National Academy of Sciences,1984,81: 8014-.
The overall KASP reaction was 5. mu.L, containing 2 XKASP Master Mix 2.5. mu.L (KBS-1016-002 from LGC), KASP Assay Mix 0.07. mu.L, at a concentration of 20 ng. mu.L -1 2.43. mu.L of the template DNA of (1);
wherein, the preparation method of KASP Assay Mix of each 100 mu L is as follows: mu.L of 100. mu.M F1 primer, 12. mu.L of 100. mu.M F2 primer, and 30. mu. L, ddH of 100. mu.M universal primer 2 O is complemented to 100 mu L; the F1 primer is a sequence which is added in front of the nucleotide sequence SEQ ID NO.1 and can be combined with FAM fluorescence (the sequence combined with FAM fluorescence used in the embodiment)As shown in SEQ ID NO. 7); the F2 primer is a primer sequence obtained by adding a sequence capable of combining with HEX fluorescence (the sequence used in the embodiment and combined with HEX fluorescence is shown in SEQ ID NO. 8) in front of a nucleotide sequence SEQ ID NO. 2; specifically, the F1 primer sequence used in the IAAV5722 group is shown as SEQ ID NO.9, and the F2 primer sequence is shown as SEQ ID NO. 10.
The KASP reaction program was: the first step is as follows: 15min at 94 ℃; the second step is that: reducing the temperature by 0.6 ℃ in each cycle at 94 ℃, 20s, 61-55 ℃ for 1min, and performing 10 cycles in total; the third step: a total of 26 cycles of 94 ℃, 20s, 55 ℃ and 1min were carried out.
PCR was purchased from LGC under the model number Hydrocycler 16 The water bath PCR instrument. The PCR results were analyzed by scanning with a fluorescence analyzer (from LGC, model PHERAStar plus).
During amplification, Ningmai No. 9 and Yangma 158 were added as control groups, and G allelic variation was observed with Yangma 158 and A allelic variation was observed with Ningmai No. 9. Through amplification verification, the primer IAAV5722 has a good amplification effect (figure 2), can be applied to breeding selection of Qtkw-4A, and has dominant allelic variation of G derived from Yangmai 158 and non-dominant allelic variation of A derived from Nigmai No. 9.
Further using the successfully developed KASP marker (primer IAAV5722) to perform genotyping on 139 wheat high-generation line materials, wherein the specific genotypes and phenotypic values are shown in Table 2:
TABLE 2139 test results of samples
TABLE 3 t-test results
Note: the numbers in parentheses indicate the amount of material carrying the corresponding allelic variation.
The routine t-test is carried out on the detection data of the table 2 by combining phenotype and genotype, the detection result is shown in table 3, the difference reaches a very significant level when P is less than 0.01, which indicates that G/A has a significant effect in thousand-grain weight selection, and the thousand-grain weight of the material carrying G dominant allelic variation is significantly higher than that of the material carrying A non-dominant allelic variation.
Sequence listing
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<120> wheat grain weight-related KASP primer group and application thereof
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agctgcaacc catcctct 18
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agctgcaacc catcctcc 18
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<213> Artificial Sequence (Artificial Sequence)
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gaaggtcgga gtcaacggat tagctgcaac ccatcctcc 39
Claims (3)
1. The application of a group of KASP primer groups related to wheat grain weight in detecting wheat grain weight is characterized in that the KASP primer groups comprise an F1 primer with a nucleotide sequence shown as SEQ ID NO.9, an F2 primer with a nucleotide sequence shown as SEQ ID NO.10 and a universal primer with a nucleotide sequence shown as SEQ ID NO. 3; the grain weight of the wheat sample carrying the G dominant allelic variation is higher than that of the wheat sample carrying the A non-dominant allelic variation;
the wheat is a recombinant inbred line group constructed by taking Ningmai No.9 and Yangmai 158 as parents.
2. Use according to claim 1, characterized by the following steps: preparing a PCR detection system by using the KASP primer group, carrying out PCR amplification on the DNA of the sample wheat, taking Yangmai 158 and Ningmai 9 as a reference, and then carrying out genotyping on the PCR amplification product of the sample wheat by using a fluorescence analyzer; the grain weight of wheat samples carrying the G dominant allelic variation is higher than that of wheat samples carrying the A non-dominant allelic variation.
3. The use of claim 2, wherein the PCR detection line is 5 μ L comprising 2.43 μ L of template DNA at a concentration of 20ng/μ L, 2.5 μ L of 2 XKASP Master Mix, 0.07 μ L of KASP Assay Mix;
wherein, the preparation method of KASP Assay Mix of each 100 mu L is as follows: 100 μ M of the F1 primer 12 μ L, 100 μ M of the F2 primer 12 μ L, 100 μ M of the universal primer 30 μ L, ddH 2 O is complemented to 100 mu L;
the PCR amplification procedure was: the first step is as follows: 15 min at 94 ℃; the second step is that: reducing the temperature by 0.6 ℃ in each cycle at 94 ℃, 20 s, 61-55 ℃ for 1 min, and performing 10 cycles in total; the third step: a total of 26 cycles of 94 ℃, 20 s, 55 ℃ and 1 min were carried out.
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